Chick high-density culture chondrocytes synthesize cartilage-specific proteoglycans with much structural similarity to the proteoglycans made by cartilage in vivo. Such cultures can be maintained in a defined medium formulated in this laboratory in which chondrogenesis occurs without the addition of serum. The proteoglycans synthesized by the chondrocytes in the presence of defined medium are of a cartilage-specific structure but differ in some aspects from the proteoglycans made in serum-containing medium. While their buoyant density, ability to aggregate with hyaluronic acid, and keratan sulfate chain size are unchanged, the proteoglycans synthesized in defined medium have altered chondroitin sulfate chains. This chondroitin sulfate is of significantly larger size and has a different sulfation pattern relative to that produced in serum-containing medium. The larger size of the chondroitin sulfate results in a larger monomer size of the defined medium proteoglycans. These differences have implications about the regulation of the structure of chondroitin sulfate proteoglycans.